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THE EFFECT OF THE SUSPENSOR AND GIBBERELLIC ACID ON
PHASEOLUS VULGARIS EMBRYO PROTEIN SYNTHESIS
by
Ellen Deloy Walthall, B.S.
A THESIS
IN
BOTANY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
Chairman of the CommitJt§fe
Accepted
Dean ot Ithe GrAduate School ot Ithe Gry
December, 1982
I n J ; ACKNOWLEDGEMENTS
,10. if I'' 6p' This paper is dedicated to the many people who helped and encour
aged me in the writing of this thesis. There is not enough room to
credit all but a special few made this possible and they follow:
Tom, whose tolerance, guidance, charity and special brand of
temper shaped me and the thesis.
Karla, Chris, Andy, Chris, Jenifer, and many others who lifted
me out of the depths of disappointment so many times.
Tom T. and Bor-Shyue H. who allowed me to do their work with my
mind elsewhere and then gave me the use of their equipment.
Joe G. and Ray J. who listened when I was afraid and worried.
To all my family for their support and helping me to "get away
from it all ."
Lynn, who did not understand my drive and motives but in spite
of the lack of understanding stayed by me.
And, a host of others.
Thank you.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS 11
ABSTRACT ^
ABBREVIATIONS ^^
LIST OF ILLUSTRATIONS vii
I. INTRODUCTION 1
II. METHODS AND PROCEDURES 4
Plants 4
Embryo Culture 4
Determination of Optimum Scurose Concentration 4
Protein Labeling 4
Exogenously Applied Gibberellic Acid 5
Acrylamide Electrophoresis 6
III. RESULTS 7
Determination of an Optimal Sucrose Concentration . . . 7
Effect of the Suspensor on Protein Synthesis and Content. 8
0.2 mm Embryos
Protein Quantity 8
Protein Synthesis 9
0.5 mm Embryos
Protein Quantity 10
Protein Synthesis 11
Effect of Gibberellic Acid (GA.) Concentration on
Protein Quantity and Protein Synthesis 12
0.2 mm Embryos
Protein Quantity 12 iii
TABLE OF CONTENTS (CONTINUED)
Protein Synthesis 13
0.5 mm Embryos
Protein Quantity 14
Protein Synthesis 15
IV. DISCUSSION 16
LIST OF REFERENCES 22
IV
ABSTRACT
In this thesis the role of the suspensor and gibberellic acid in
Phaseolus vulgaris embryo protein content and synthesis was examined.
The plant embryo exists in a very specialized environment and this
environment must be maintained in tissue culture for continued nor
mal embryonic development. Optimum sucrose concentrations for cult-ure
of 0.2 mm and 0.5 mm embryos in Gamborg B5 medium were determined
to be 12^ and 6^ respectively. Protein content and synthesis of
various culture combinations of these embryos and their suspensors
were examined by polyacrylamide electrophoresis. Two-tenths millimeter
embryos required an attached suspensor for maximum protein content.
Virtually all protein synthesis was dependent upon an attached suspen
sor. Maximum protein quantity and synthesis in 0.5 mm embryos were
observed when the embryo was cultured attached to the suspensor.
Protein levels decreased when it was cultured detached from or without
_7 the suspensor. Gibberellic acid of 10 M elicited the same protein
35 diversity and greater S -methionine incorporation than the attached
suspensor in 0.2 mm embryos. Five-tenths millimeter embryos did not
appear to be differentially responsive to various gibberellin concen
trations. All of the Gl storage protein subunits and the 32 and 3^
kD G2 storage protein subunits were observed in 0.2 mm embryos. The
0.5 mm embryos in gibberellin had all of the Gl and G2 storage pro
tein subunits.
V
ABBREVIATIONS
E = Embryo cultured in the absence of the suspensor
E/E-S = Embryo of embryo cultured attached to the suspensor
E/E+S = Embryo of embryo cultured with a detached suspensor
GA = Gibberellic Acid
kD = KiloDaltons
S.A. = Specific Activity
S/E-S = Suspensor of embryo cultured attached to the suspensor
S/E+S = Suspensor of embryo cultured with a detached suspensor
VI
LIST OF ILLUSTRATIONS
Figure lA. Effect of sucrose concentration on 0.2 mm embryo elongation after seven days of culture.
B. Effect of sucrose concentration on 0.5 mm embryo elongation after seven days of culture.
Figure 2A. Effect of the suspensor on protein quantities (jig per organ) of 0.2 mm embryos.
B. Coomassie Blue stained 10% SDS polyacrylamide slab gel demonstrating the suspensor's effect on specific protein quantities and distribution.
C. Fluorograph of the gel shown in B.
Figure 3A. Effect of the suspensor on protein quantities (yg per organ) of 0.5 mm embryos.
B. Coomassie Blue stained 10% SDS polyacrylamide slab gel demonstrating the suspensor's effect on specific protein quantities and distribution.
C. Fluorograph of the gel shown in B.
Figure 4A. Effect of gibberellic acid concentrations on protein quantities (yg per organ) of 0.2 mm embryos.
B. Coomassie Blue stained 10% SDS polyacrylamide slab gel demonstrating gibberellic acid's effect on specific protein quantities and distribution.
C. Fluorograph of the gel shown in B.
Figure 5A. Effect of gibberellic acid concentrations on protein quantities (yig per organ) of 0.5 mm embryos.
B. Coomassie Blue stained 10% SDS polyacrylamide slab gel demonstrating gibberellic acid's effect on specific protein quantities and distribution.
C. Fluorograph of the gel shown in B.
Vll
I. INTRODUCTION
The function of the angiosperm suspensor was classically thought
to be purely mechanical, i.e., pushing the embryo into the nutrient-
rich endosperm (Maheshwari, 1950). However, Gunning and Pate (1969)
pointed out that the wall projections found on suspensor cells of
numerous angiosperms suggested a transfer (transport) function for
this organ due to the increased surface area.
Cionini et al. (1976) found that in Phaseolus coccineus heart-
shaped embryos of less than 5 mm length (suspensor excluded), sus
pensor removal caused a decrease in the percentage of embryos sur
viving tissue culture. Survival percentages ranging from 44% when
grown intact (15 days) to 7% when grown without the suspensor were
obtained for 0.5 mm embryos. Larger embryos (2.0 mm to 5.0 mm) had
up to a 30% decrease in survival when cultured in the absence of the
suspensor. Clearly the suspensor has some effect on the embryo;
this study will -attempt to determine the suspensor's effect and how
it influences the embryo by observing the embryo protein quantity
and synthesis.
In a white-seeded variety of the runner bean, Phaseolus coccineus,
the suspensor had 30 times the gibberellin activity as the embryo at
the heart-shaped stage (Alpi et al., 1975). At the cotyledonary stage,
the level of gibberellin activity in the suspensor dropped while tlie
gibberellin activity in the embryo rose until the two tissues had
approximately the same levels of activity. At this time, the overall
gibberellin level in the embryo had increased about 10 times above
that at the heart stage.
1
2
Gibberellic acid has been shown to initiate increases in the
activity of several enzymes including a-amylase (Hooley, 1982; Varner,
1964; MacLeod and Millar, 1962; Paleg, 1960), g-glucanase (Briggs, 1963;
MacLeod and Millar, 1962), phosphatase (Briggs, 1963), and ribonuclease
(Hooley, 1982; Kapoor, 1981; Chrispeels and Varner, 1967). Kapoor
(1981) found that GA stimulated RNAase activity two- to three-fold in
germinating seeds and that this enhancement was due to de_ novo syn
thesis. Varner and Ram Chandra (1964) demonstrated that in germinating
barley seeds (Hordeum -vulgare var. Himalaya), a gibberellic acid-
dependent increase in o-amylase activity required de^ novo protein syn
thesis. Further work on H . vulgare (Varner et al., 1965) showed
the necessity of RNA synthesis for this increase in a-amylase activity.
Gibberellic acid also has been shown to enhance RNA synthesis in
isolated pea nuclei. These newly synthesized RNAs differed both in
base sequence and size from the RNAs of untreated nuclei (Johri and
Varner, 1967).
The high gibberellin activity in the suspensor and the ability of
gibberellin to enhance RNA and protein synthesis in other systems
(Hooley, 1982; Kononowicz et al., 1982; Martin and Northcote, 1982;
Kapoor, 1981; Sawhney et al., 1977; Srivastava et al., 1975) make it
reasonable to examine whether the suspensor may play a physiological
role (i.e., regulation of protein synthesis) during Phaseolus embryo
development. If it can be established that the suspensor has such a
role, then I wish to determine whether GA, can be employed to mimic the
suspensor in regulating embryo protein content and synthesis.
In this thesis the effect of the suspensor on protein content
3
and synthesis during two stages of embryo development will be examined.
The following questions will be addressed:
(1) Does the presence or absence of the suspensor affect
embryo protein content or synthesis?
(2) If there is an effect, could the gibberellin activity in
the suspensor be responsible for part or all of the sus
pensor 's effect on the embryo?
II. METHODS AND PROCEDURES
Plants
Phaseolus vulgaris var. Taylor's Horticultural (Courtesy of
Asgrow Seed Company) seeds were grown in seven inch pots in a growth
chamber at 24 * 2°C. Light intensity ranged from 284 to 384 micro-
2 einsteins/m -sec on a 12 hour photoperiod. Pods were harvested when
1 to 4 centimeters in length.
Embryo Culture
The pods were surface sterilized with absolute ethanol, opened
and the embryos excised with sterile tungsten needles and cultured in
10 yl of sterile Gamborg's B5 medium (Gamborg, 1968) in sterile Falcon
Microtest Plates and cultured at 27 * 2 C on a 12 hour photoperiod.
Determination of Optimum Sucrose Concentration for Embryo Culture
Five-tenths mm embryos (suspensor excluded) were cultured in
Gamborg's B5 medium containing 2, 4, 6, 8, 10, 12, and 14% sucrose
for 7 days to determine the sucrose concentration yielding optimum
growth. Embryos 0.2 mm long were cultured in 6, 8, 10, 12, 14, and
16% sucrose under the same criteria as for the 0.5 mm embryos. Length
measurement of both sizes of embryos were made daily.
Protein Labeling
Five-tenths mm embryos were cultured in Gamborg's B5 medium
(Gamborg, 1968) containing 6% sucrose and 0.2 mm embryos in
medium containing 12% sucrose for 48 hours with (a) the suspensor
5
attached, (b) the suspensor present but unattached and (c) in the
absence of the suspensor. Fifteen of the 0.5 mm embryos or forty of
the 0.2 mm embryos of each state were combined for labeling the last
hour of culture in 20 pi of Gamborg's B5 medium with the appropriate
35 sucrose concentration. One yCi of S -methionine was used for each
three 0.5 mm embryos and each eight 0.2 mm embryos (S.A. ranged from
1.21 iiCi/jil to 5.59 yCi/yl).
Exogenously Applied Gibberellic Acid
Three 0.5 mm and 40 0.2mm embryos were cultured without the
suspensors in 10 yl of medium containing appropriate sucrose concen-
—R —7 —fi trations and gibberellic acid (Sigma Chemical) at 10~ , 10~ , 10 ,
-5 -4 10 , and 10 M concentrations for 48 hours. Newly synthesized
35 proteins were radioactively labeled (S -methionine) during the last
hour of culture in 20 yl of Gamborg's B5 medium with the appropriate
sucrose and gibberellin concentrations. The medium also contained
35 1 yCi of S -methionine (S.A. ranging from 1.21 jiCi/yl to 5.59 yCi/yl)
for each three 0.5 mm embryos and each eight 0.2 mm embryos.
Protein Determination
Protein concentrations were determined by homogenizing three 0.5
mm embryos or suspensors or thirty 0.2 mm embryos or suspensors in
200 jil of water. Samples were then centrifuged at 7000 X g in a Fisher
Model 59 centrifuge for 20 minutes at room temperature. Assays were
performed in duplicate on 90 yl of this homogenate. Protein concen
tration was determined according to the method of Bradford (1976) using
the Bio-Rad Protein Determination Kit.
6
Acrylamide Electrophoresis
Embryos and suspensors (numbers reported in previous sections)
were homogenized separately in 100 yl of sample buffer (LeStourgeon
and Beyer, 1977) and centrifuged at 7000 X g in a Fisher Model 59
Centrifuge for 20 minutes at room temperature. The supernatants
were electrophoresed on 10% SDS polyacrylamide slab gels (LeStourgeon
and Beyer, 1977). The gels were stained with coomassie blue G250,
destained in 7-10% acetic acid, impregnated with Enhance (New England
Nuclear) and dried. Fluorography was perfo-rmed as described in
Bonner and Laskey (1976) using Kodak X-R5 film. Exposure was from 1
to 30 days at -70 C. The standards were bovine serum albumin (MW
66,000 daltons), ovalbumin (MW 45,000 daltons), trypsinogen (MW 24,000
daltons), g-lactoglobulin (MW 18,400 daltons), and lysozyme (MW 14,300
daltons), all of which were purchased from Sigma Chemical.
RESULTS
Determination of an Optimal Sucrose Concentration
Many plant embryos, when cultured, do not complete the remaining
developmental stages but instead germinate (Long et al., 1981). This
precocious germination has been shown to be retarded or completely
blocked by a decrease in osmotic potential (higher sucrose concen
tration) - The smaller the embryo the more negative the osmotic
potential of the culture medium must be for proper developmental pro
gression (Yeung and Brown, 1982; Rietsema et al., 1953).
To determine the optimal sucrose concentration necessary for
normal developmental appearance, 0.5 mm embryos were cultured in
Gamborg's B5 media containing 2, 4, 6, 8, 10, 12, and 14 percent
sucrose (Figure IB);0.2 mm embryos were cultured in media containing
6, 8, 10, 12, 14, and 16 percent sucrose (Figure lA). Mean percent
increase in length was determined after 7 days in culture.
The 0.2 mm embryos, being smaller, were tested over a higher
concentration range than were the 0.5 mm embryos. Although the
differences in 0.2 mm embryos cultured in various sucrose concen
trations between 6 and 16% were not significant (standard errors
overlapped), 12% sucrose medium yielded the greatest mean percent
increase in length. Embryo color and cotyledon shape remained normal
at this sucrose concentration and it was selected for all subsequent
cultures of 0.2 mm embryos.
Five-tenths mm embryos cultured in 2% sucrose medium formed
callus, usually at the point of embryo-suspensor attachment. This
accounts for the extremely large increase in length recorded
7
;o ;o icM 3C3> ICM sO i O
iiT)
=C0
Figure 1. Effect of sucrose concentration on embryo elongation after seven days of culture. Mean percent increase is reported for each concentration. A. —•— 0.2 mm embryos; B. —•- 0.5mm embryos. Embryos were cultured attached to suspensors but only embryo length was measured (n = IC
250-
200-
150.
100.
50-
6 8 10 12
PERCENT SUCROSE
14 16 18
10
250-
200.
150.
H 100-
50-
6 8 10 12
PERCENT SUCROSE
14 16 IS
11
for embryos cultured at this concentration of sucrose. There was a
sharp drop in mean percent embryo elongation when embryos were cul
tured in 4% sucrose Gamborg's medium. The embryos cultured in 6%
sucrose medium had a slightly higher mean increase in embryo length.
All successive concentrations (8, 10, 12 and 14%) steadily declined
in mean percent embryo elongation until 14% sucrose Gamborg's B5
medium had only slightly more than a 50% increase in length. Six
percent sucrose Gamborg's B5 medium was chosen for 0.5 mm embryo
cultures. Embryos cultured in this medium, in addition to having the
greatest mean percent increase in embryo length other than 2% sucrose
(which formed callus), also most closely resembled the freshly excised
embryos in color and cotyledon shape.
Effect of the Suspensor on Protein Synthesis
In order to determine whether the suspensor has an effect on
embryo protein content and synthesis, 0.5 mm and 0.2 mm embryos were
cultured for forty-eight hours with the suspensor attached to the
embryo, with the suspensor detached from but cultured in the same drop
of medium as the embryo, or in the absence of the suspensor. At the end of
the culture period, total protein quantity (Bradford, 1976) was deter
mined in both the embryo and the suspensor. Protein synthesis was
35
examined by incubation of embryos in S -methionine followed by elec
trophoresis and fluorography.
0.2 mm Embryos
Protein Content
Of the cultured 0.2 mm embryos, maximum protein quantities were
12
observed in both embryos and suspensors when they were cultured
attached (0.190 * 0.031 yg, 0.135 ± 0.020 yg; embryo and suspensor,
respectively)(Figure 2A). These levels were significantly lower than
that of fresh tissues (0.359 ^ 0.076 yg, embryo; 0.293 * 0.071 ;ig,
suspensor). Severance of the suspensor from the embryo caused a
further reduction in protein quantity in the embryo (0.150 * 0.032 yg,
detached; 0.190 * 0.031 pg, attached) but had no affect on the suspen
sor (0.133 - 0.014 yg, detached; 0.135 ± 0.020 ;ig, attached). Two-
tenths milimeter embryos cultured in the absence of the suspensor
showed the least amount of protein (0.130 - 0.024 _yg) of the embryos
examined.
When proteins of the 0.2 mm embryos cultured with the suspensor
attached (E/E-S) were examined on SDS polyacrylamide gels (Figure 2B),
both the nvmiber of bands present on the gel and the staining intensity
of these bands were greater than when 0.2 mm embryos were cultured
with the suspensor detached (E/E+S). The 0.2 mm embryo cultured in
the absence of the suspensor (E) had the fewest bands and the lowest
staining intensity of any of the 0.2 mm embryos cultured. The sus
pensor banding pattern appears the same whether the suspensor is
cultured attached to the embryo (S/E-S) or severed from the embryo (S/E+S),
Protein Synthesis
The major proteins synthesized in both embryos and suspensors
cultured under the various experimental conditions appear to remain
relatively constant. There are marked differences in the amount of
incorporation which corresponds to the total protein content.
Radioactive amino acid incorporation (Figure 2C) in 0.2 ran embryos
13
15
B
^^n^
A E' S'
E-S E-S E
E-S S
E-S E
E+S S
EfS
16
(E) and suspensors (S) peaked when the tissues were cultured attached
(E-S). This incorporation was radically reduced when the embryo (E)
and suspensor (S) were detached (E+S) or the embryo was cultured
alone (E). At the early stage (0.2 mm), protein bands corresponding
to 60, 47, 43, and 36 kD molecular weight are the major proteins
being synthesized in embryos cultured attached to their suspensors.
Except for the protein of 60 kD, whose intense synthesis is observed
in embryos cultured detached from their suspensor, the synthesis of
these proteins is drastically reduced or absent in embryos cultured
either detached from their suspensors or alone. Peptides which
migrate at the same molecular weight as these newly synthesized
peptides were observed in the Coomassie Blue stained gel of proteins
extracted from all cultured embryos. The bands at 43 and 36 kD
stained in embryos cultured attached to their suspensors and in
embryos cultured with a detached suspensor but not in embryos
cultured alone. In the fluorograph this band was synthesized
only in the embryo which was cultured attached to its suspensor.
0.5 mm Embryos
Protein Content
The 0.5 mm embryos and suspensors had maximum quantities of
protein when cultured attached to each other (2.508 ± 0.553 yg, 1.882 *
0.315 yg; embryo and suspensor, respectively. Figure 3A). These quan
tities were not significantly different from the protein quantities
of fresh tissues (3.251 ± 0.398 >ig, 2.290 * 0.214 yg; embryo and sus-
17
Figure 3A. Effect of the suspensor on protein quantities (>ig per organ) of 0.5 mm embryos. E/E-S, signifies the embryo when cultured attached to the suspensor; S/E-S, signifies the suspensor when cultured attached to the embryo; E/E+S, signifies the embryo when cultured with but detached from the suspensor; S/E+S, signifies the suspensor when cultured with but detached from the embryo; and E signifies the embryo cultured alone. For each condition, n = 5. Standard error is reported in each case. Organs were cultured 48 hours in Gamborg's B5 medium with 6% sucrose.
B. Coomassie Blue stained 10% SDS polyacrylamide slab gel demonstrating the suspensor's effect on specific protein quantities and distribution. The numbers to the right, represent molecular weights of standard proteins in 10 Daltons. n = 15. (—) mark the calculated mobilites of the Gl and G2 proteins' subunits molecular weights (53, 49, 47, 43 and 34, 32, 30 kD, respectively). (•) mark the proteins refered to in the thesis.
C. Fluorograph of the gel shown in B. E' was exposed 5 days, the remainder of the wells were exposed for 9 days.
18
4.0 -
3 . 0 -
i 2 . 0 .
1 . 0 .
E Fresh
E E-S
E E + S
E Alone
S Fresh
S
E-S S
E + S
19
B
- 6 6
—45
20
pensor, respectively). Five-tenths mm embryos cultured with
but detached from their suspensors had a lower protein quantity
(1.910 ^ 0.252 yg) than those cultured attached to the suspensor
(2.508 - 0.553 jag). The embryos cultured in the absence of the sus
pensor showed a sharp reduction in protein (1.044 - 0.304 yg) which
was significantly lower than that of fresh embryos or those cultured
in the presence of the suspensor.
Suspensors cultured detached from embryos had less than half the
protein of the suspensors cultured while still attached to the embryo
(0.723 - 0.194 yg, 1.882 ± 0.315 yg, respectively; Figure 3A).
An examination of the Coomassie Blue stained gel of proteins ex
tracted from 0.5 mm embryos indicated that in general there is a
decrease in stain intensity in all of the bands on the gel in the
following order: E/E-S > E/E+S > E (Figure 3B). This also is true
of any given band from lane to lane.
Protein Synthesis
Embryos (E) cultured attached suspensors (E-S) show intense
35 labeling with S -methionine (Figure 3C). Embryos (E) cultured with
detached suspensors (E+S) show lower levels of incorporation, while
embryos (E) cultured without suspensors had lowest levels of radio-
labeling. Figure 3C shows two proteins at about 30-32 kD being syn
thesized in embryos under all culture conditions. A very faint band
at 30,000 daltons was observed in 0.2 mm embryos (embryos of embryos
cultured attached to suspensors) but not in embryos cultured detached
from or in the absence of the suspensor (Figure 3C) . The major proteins
being synthesized in 0.5 mm embryos remain those at molecular weights
21
of 60, 53, 47, 43 and 36 kD which were observed in 0.2 mm embryos.
Unlike the 0.2 mm embryos in which the synthesis of these proteins
was observed only, or to the greatest extent in embryos cultured
attached to their suspensors, the 0.5 mm embryos synthesized these
proteins under all of the culture conditions examined.
Effect of Gibberellic Acid (GA ) Concentration on Protein Quantity
and Protein Synthesis
It has been previously demonstrated that the suspensor has a
high level of gibberellic acid activity. Results in the previous
section indicate that the presence of the suspensor attached to the
35 embryo positively influences S -methionine incorporation into the
embryo's total protein. The role of gibberellins in the regulation
of protein content and protein synthesis in Phaseolus embryos was
examined. Embryos were cultured without their suspensors in con-
-4 -8 centrations of gibberellic acid of 10 M to 10 M for 48 hours.
Total protein content was determined and protein synthesis was
assayed as previously described.
0.2 mm Embryos
Protein Quantity
Embryos cultured in 10~ M gibberellic acid had slightly higher
protein levels than those of embryos cultured without exogenous gib
berellin but significantly lower levels than those of fresh embryos
(0.146 ± 0.013 Jig, 0.130 i 0.024 yg, 0.359 ± 0.076 >ig, respectively)
(Figure 4A). Embryos cultured in 10~ M gibberellic acid had protein
quantities (0.203 * 0.024 )ig) which were significantly greater than
22
Figure 4A. Effect of gibberellic acid concentrations on protein quantities (yg per organ) of 0.2 mm embryos. Molar concentrations of GA, in which embryos were incubated are labeled at the bottom of the appropriate histogram. In each case standard error is reported (n = 5).
B. Coomassie Blue stained 10% SDS polyacrylamide slab gel demonstrating gibberellic acid's effect on specific protein quantities and distribution. The concentrations are labeled at the bottom of each well. (n = 40) The numbers to the right represent molecular weights of standard proteins in 10 Daltons. (-) mark the calculated mobilities of the Gl and G2 proteins' subunits molecular weights (53, 49, 47, 43 and 34, 32, 30 kD, respectively). (•) marks the specific peptides synthesized in response to GA .
C. Fluorograph of the gel shown in B. Exposure was for 4 days.
23
0.5-1
0.4-
0.1-
O z
0.2-
o.i-
10 10 10 10 10 -8
E Fresh E-S Alone
24
B
<«MM)>fi> ^'-ififm • 4 | M V « »
Hjl ^yi ' lij^ ^^
—66
" — 45
— 24
— 1 8
- 6 6
; —45
it It ^ — 24
—1 8
10 10^ 10~" 10^' 10 / .^-8
25
embryos cultured alone and without exogenous gibberellin. Two-
tenths mm embryos cultured in gibberellic acid showed maximal protein
— fi 7
quantities when cultured in 10~ M (0.287 - 0.021 yg) or 10~ M
(0.288 i 0.034 yg) gibberellin (Figure 4A). These quantities are
significantly higher than those found in embryos cultured attached to
suspensors in medium lacking gibberellin (0.130 - 0.024 jig), and are
slightly lower but statistically indistinguishable from those of —8
freshly excised uncultured embryos (0.359 ^ 0.076 jig). The 10 M GA,
caused a response slightly lower than 10 M or 10 M, giving 0.266 -
0.038 yg of protein. When comparing these quantities with those ob
served in 0.2 mm embryos cultured with the embryo and suspensor
—6 —7 —8 attached, 10 , 10 and 10 M GA_ gave significantly higher protein
quantities (Figure 4A). It is interesting to note that while the pro
tein content of 0.2 mm embryos cultured attached to the suspensor
(0.190 * 0.031 yg) was significantly lower than fresh tissue (0.359 *
0.076 yg) , the protein content of those embryos cultured without their
—6 —7 —8
suspensors in 10 , 10 or 10 M gibberellic acid was not signifi
cantly different from fresh tissue.
The Coomassie Blue stained gel of protein extracted from embryos
cultured in various GA_ concentrations (Figure 4B) demonstrates that
although the banding pattern for each well on the gel is identical,
-4 the staining intensity indicating protein quantity increased from 10
—7 —8 to 10~ M and then decreased at 10 M gibberellic acid.
Protein Synthesis
-4 -5 Figure 4C shows that 0.2 mm embryos cultured in 10 M and 10 M
GA had relatively low levels of radioactive incorporation although
26
substantially higher than that of embryos cultured without exogenous
gibberellic acid (Figure 2C, E). The 10~^ and 10~^ M GA -treated
embryos had high levels of label incorporation with the 10~^ M GA -
treated embryos having only slightly lower levels of incorporation
although higher than that of embryos cultured with the suspensor.
In addition to the general increases noted above, increased syn
thesis of specific proteins in the range of 65, 49 and 34 kD are seen
at the lower gibberellin concentrations. The 65 kD band appears in
—fi — 7 ft all concentrations but is enhanced in 10 ,10 and 10~ M GA . A
—7 —8 protein band of about 34 kD appears in the 10 and 10 M gibberellic
acid concentrations (Figure 4C).
0.5 mm Embryos
Protein Quantity
—8 Five-tenths mm embryos cultured in 10 M gibberellin (Figure 5A)
showed the same protein concentration as that of the freshly excised
uncultured embryos (3.222 ± 0.388 ug; 3.251 ± 0.398 ug, respectively),
both of which were significantly higher than embryos cultured alone
without gibberellin (1.044 * 0.304 ug). Concentrations of GA3 higher
—8 than this level had quantities slightly lower than 10 M GA_-treated
embryos and fresh tissue but significantly higher protein levels than
found in embryos cultured in the absence of the suspensor and without
any exogenous gibberellin (10 M, 2.543 - 0.363 ug; 10~ M, 2.406 ±
0.282 ug; 10"^ M, 2.148 - 0.317 ug; and 10~^ M, 2.660 * 0.298 ug). All
concentrations yielded protein quantities which were indistinguishable
from the protein quantities of the embryo cultured attached to the sus
pensor (E/E-S).
27
29
B
— 6 6
•—4 5
10 10 10 10"' 10
30
Protein Synthesis
The general pattern and intensity of amino acid incorporation is
the same for embryos cultured in gibberellic acid concentrations
-4 -8 between 10 M and 10 M. Specific proteins in the ranges of 30,
49 and 65 kD are synthesized in response to low gibberellic acid con
centrations and are absent at concentrations 10 M and above.
DISCUSSION
The role of the angiosperm suspensor in the development of the
embryo has not been thoroughly examined. Based on the presence of
finger-like projections between the suspensor and the sterile seed
tissue (Schnepf and Nagl, 1970) and on the observation that these
projections are found in plant cells with a transfer function (Gunning
and Pate, 1969), it has been postulated that the suspensor may serve
the function of transfering compounds synthesized either in the sus
pensor or in other parts of the seed or plant t o the developing embryo
(Clutter and Sussex, 1968; Brady, 1973). If the suspensor plays such
a role, then it must do so early in the development of the embryo because
in the Phaseolus -vulgaris embryo the suspensor reaches its maximum
size at the late heart stage and then begins to degenerate (Walbot
et al., 1972). Gibberellin, a plant hormone which has been shown to
play a role in the development of several plant embryos (Kefford and
Rijven, 1966; Dure and Jensen, 1957), has been demonstrated to be
present in high quantities in the suspensor of early Phaseolus embryos,
and its concentration decreases in later development (Alpi et al.,
1975). Therefore, the effect of the suspensor in vitro and of gibber
ellins on protein synthesis and content in two early stages of embryo-
ogenesis in Phaseolus vulgaris has been examined.
The plant embryo exists in a very specialized environment, and
this environment must be maintained in tissue culture for continued
normal embryonic development (Raghavan, 1976). In Phaseolus (Yeung
and Brown, 1982; Smith, 1973), as with several other plant embryos
31
32
(See Raghavan, 1976 for a review) , the osmotic potential of the endo
sperm surrounding the embryo and that of the embryo itself is high
early in development and decreases as development proceeds. The
results presented in this thesis demonstrate that for 0.2 mm Phaseolus
•vulgaris 4 days post-anthesis early heart stage embryos 12% sucrose in
the culture medium produced maximal growth without either precocious
germination or callus formation. Three days later at seven days post-
anthesis , the sucrose concentration at which maximal normal develop
ment was observed dropped to 6% sucrose. These results are strikingly
similar to those obtained for Datura (Rietsema et al., 1953) and
Hordeum (Norstog, 1961).
The suspensor clearly affects protein quantities. With both
embryo sizes, the presence of an attached suspensor resulted in the
highest quantities of protein. When the suspensor was severed, both
sizes of embryo showed a decrease in protein content. A further
decrease was observed when the suspensor was removed.
Protein synthesis in 0.2 mm cultured embryos was strongly affected
by the attached suspensor. Amino acid incorporation virtually ceased
when 0.2 mm embryos were cultured with a severed suspensor or without
the suspensor. Five-tenths mm embryos were not as dependent upon sus
pensor attachment as were those of 0.2 mm. Removal of the suspensor
from these embryos, moderately reduced protein synthesis. Even 0.5
35
mm embryos cultured without suspensors showed substantial S -methio
nine incorporation.
Low gibberellin concentrations almost doubled the protein content
of 0.2 mm embryos cultured without their suspensors. The protein content
33
of embryos cultured with attached suspensors (E/E-S) was significantly
lower than that of fresh embryos, but these quantities are statistically
indistinguishable from fresh tissues. If the suspensor supplied the
developing embryo with gibberellin, and as it has been demonstrated GA,
affects the protein content of the embryo, a large culture medium vol
ume (10 yl) without any exogenous GA would dilute the gibberellin of
the suspensor so that even with the embryo attached, the resultant
protein quantities would be lower than that attained ^^ vivo. If one
assumes that the major route of the GA, to the embryo is from the
suspensor directly into the embryo and not into the surrounding
tissue or through the endosperm, then the difference in protein between
the embryos cultured attached to the suspensor and those cultured
separated from their suspensor may be explained.
Cionini et al. (1976) found that these same low gibberellin con-
—8 —6 centrations (10 M to 10 M) which enhance protein content of 0.2 mm
embryos cultured o^ vitro also Increased survival of heart-stage embryos
of Phaseolus coccineus cultured in the absence of the suspensor.
Five-tenths millimeter embryos cultured in all gibberellin concen
trations examined had a higher quantity of protein than that observed
in embryos cultured in the absence of the suspensor and were indistin
guishable from E/E-S. The 10~ M and 10~ M gibberellin-treated
—7 —8 embryos had significantly lower protein quantities than 10 or 10 M
gibberellin-treated or freshly excised embryos.
The synthesis of several bean embryo proteins has been closely
examined (Murray and Crump, 1979; Sun et al., 1978; Barker et al.,
1976; and Derbyshire and Boulter, 1976). Those proteins which have
received the most attention are the storage proteins. These have been
34
divided into two major groups. The Gl (glycoproteins I) proteins have
been extensively examined by the laboratory of T. C. Hall (Brown et al.,
1981; Sun et al., 1981; Brown et al., 1980; Ma et al.. 1980; Mutschler
et_al., 1980; Hall et al., 1978; Sun et al., 1978; Stockman et al.,
1976; Sun and Hall, 1975; and McLeester et al., 1973) in the cultivar
Tendergreen, and less so by the Sussex laboratory (Sussex, personal
communication) in the cultivar Taylor's Horticultural. This latter
cultivar was used in the present study. Although there is some dis
agreement as to the genetics of this system, these authors do agree
that the major subunits of the Gl proteins have molecular weights of
53, 47 and 43 kD and that a minor component with a molecular weight
of 49 kD also exists but is not always expressed. The G2 (glycopro
teins II) proteins have also been examined by the Hall laboratory
(Mutschler et al. , 1980; Sun and Hall, 1975; and McLeester et al., 1973).
These proteins are composed of peptides separating into 3 bands on
polyacrylamide gels with molecular weights of 30, 32 and 34 kD
(McLeester et al., 1973).
Most of the work done on these proteins up to the present has
concentrated on the appearance of stained bands on polyacrylamide gels
as an indication of when these proteins were synthesized. The one
exception (Sun et al., 1978) indicated that the synthesis of these
proteins began at the 7 mm bean stage (at 12 days post-anthesis) . The
work reported here demonstrates that at the 0.2 mm stage embryo (2.0 mm
bean), 4 days post-anthesis, all of the Gl peptides are synthesized
in embryos cultured forty-eight hours attached to their suspensors,
but their synthesis in embryos cultured in the presence of but detached
35
from their suspensor or in embryos cultured without their suspensor
is much reduced. These same peptides were observed to be intensely
synthesized by embryos cultured in gibberellin in the absence of their
suspensors at all of the concentrations examined. This may indicate
the synthesis of all of these proteins is under the regulation of GA,.
The G2 proteins show a similar response. The 32 kD subunit is
synthesized in 0.2 mm embryos cultured attached to their suspensor but
not in embryos cultured under other culture conditions. In 0.5 mm
embryos both the 32 kD subunit and a 30 kD subunit are synthesized.
This same synthetic pattern (30 and 32 kD) is observed in 0.2 mm embryos
in all concentrations of GA, examined. At low levels of gibberellic
acid the synthesis of a third peptide of 34 kD is observed. The syn
thesis of this band is most intense at 10 M GA^ but is substantially
—fi —8
reduced at both 10 and 10 M gibberellin and is absent in GA^ con
centrations higher than 10~^ M. The 32 kD band has been demonstrated
by Hall to be the peptide most abundant in the young embryos (Sun
et al., 1978). The synthesis .of the 32 kD band is much greater than
that of the 30 or 34 kD bands and the synthesis of all of these pep
tides is most responsive to a gibberellin concentration of 10 M.
Although there are a few peptides whose synthesis appears to
be gibberellin activated, gibberellin appears to stimulate protein
synthesis in general rather than altering the amino acid incorporation
into particular protein bands. This also,-has been observed in germi
nating castor bean seeds (Martin and Northcote, 1982) and in lettuce
hypocotyls (Srivastava et al., 1975).
From these data one concludes that the 0.2 mm embryos are strongly
36
affected by the suspensor and that this effect can be mimicked by low
gibberellin concentrations. This mimicry results in the same protein
pattern as that induced by the suspensor. The 0.5 mm embryo is in
fluenced by the suspensor but is not dependent upon it for the main
tenance of protein synthesis. Equally evident .is the fact that
gibberellin does something to alleviate the lack of the suspensor.
Exactly how protein synthesis is influenced in the 0.5 mm embryos by
gibberellin is not clear. The 0.5 mm embryo is at that stage where
growth is changing from an increase in cell number to an increase in
cell size. Further, this is a time of the elegant altering of hor
mone balances (Yeung and Brown, 1982; Hsu, 1979). Free abscisic acid
(ABA) levels are 1.5 times higher in the 0.5 mm (7 days post-anthesis)
embryos than in the 0.2 mm embryos (4 days post-anthesis)(Hsu, 1979).
At later stages peaks of free ABA coincided with inhibited development
(both cell and tissue, Haddon and Northcote, 1976) and enhanced protein
synthesis. These peaks coincided with a decrease in RNA synthesis.
Haddon and Northcote (1976) also reported that gibberellic acid could
not overcome the inhibitory effect of ABA. It is quite possible that
factors other than those discussed here are involved in the 0.5 mm
embryo response. Further work is needed to determine exactly how
development is controlled and what role gibberellin plays in the
development of 0.5 mm embryos remains to be clarified.
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